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Antibody Response and Reduction in Bursa of Fabricius and Spleen Weights of Progeny of Chickens Fed PCBs
Antibody Response and Reduction in Bursa of Fabricius and Spleen Weights of Progeny of Chickens Fed PCBs SUSAN J. HARRIS, H E L E N E C. C E C I L , JOEL BITMAN AND ROBERT J. LILLIE
U.S. Department of Agriculture, Agricultural Research Service, Animal Physiology and Genetics Institute, and Nutrition Institute, Beltsville, Maryland 20705 (Received for publication February 17, 1976)
POULTRY SCIENCE 55: 1933-1940, 1976
P
1973; Britton and Huston, 1973; Cecil et al, 1974; Lillie et al, 1974, 1975; Scott et al, 1971) or injecting eggs with Aroclor 1242 or 1254 (Carlson and Duby, 1973; McLaughlin et al, 1963) reduces hatchability of chicken eggs and also reduces growth of progeny (Lillie et al, 1974).
OLYCHLORINATED biphenyls (PCBs) are persistent environmental pollutants of industrial origin which accumulate in the lipid of animal tissues. In 1971, leakage of PCBs into fish meal resulted in the contamination of feed consumed by millions of chickens. Large numbers of chickens and eggs containing excessive PCB residues were condemned (Harris, 1971; Pichirallo, 1971). Commercial chicken breeders observed decreased hatchability of eggs from flocks fed contaminated feed. Since that time, laboratory studies have shown that feeding hens 20 p.p.m. of Aroclor 1 1232, 1242 and 1248 (Briggs and Harris,
In previous studies, we characterized the PCB induced decrease in hatchability, the time of embryonic death, and the nature of the teratogenic effects occurring after feeding chicken hens PCBs (Cecil et al, 1974; Lillie et al, 1974, 1975). Most of the hatchability failure occurred during the last 2 days of
1. The PCBs are marketed in the United States under the trade name Aroclor (Monsanto) and contain a mixture of chlorinated biphenyls varying in chlorine content. Aroclor mixtures are designated by a four digit number, the last two numbers specify the percentage of chlorine in the mixture. One exception, Aroclor 1016 (41% chlorine), is a distillate of Aroclor
1242 from which the higher PCB homologs are removed. Aroclor 1016 contains 0.4% higher homologs while Aroclor 1242 contains 7% of the tetra-, pentaand hexachloro-homologs. Trade names are used solely for the purpose of providing specific information and do not imply endorsement or approval by the U.S. Government over other products not mentioned.
1933
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ABSTRACT In Experiment 1, polychlorinated biphenyls (Aroclors 1016, 1232, 1242, 1248, and 1254) were fed ad libitum to White Leghorn chickens at dietary levels of 5, 10, and 20 p.p.m. for 8 weeks. Hatchability of eggs from hens fed 10 and 20 p.p.m. of Aroclors 1232, 1242, and 1248 was reduced. The weights of the spleen and bursa of Fabricius of day-old chicks from hens fed 10 p.p.m. of Aroclors 1242 and 1248 or 20 p.p.m. of Aroclors 1232, 1242, 1248, and 1254 were less than those of control chicks. At 3 weeks of age these organ weights were still less than those of the control chicks. Body weights were similar to those of controls at hatching, but the 3 week weight gains of progeny from hens fed 20 p.p.m. Aroclors 1242 and 1248 were less than gains of control progeny. After replacement of the PCB diet with uncontaminated feed, hatchability improved within 2 weeks and by 8 weeks was similar to that of the controls. Bursa weight of the progeny was the only parameter still reduced after the hens were fed uncontaminated feed. In Experiment 2, hens were fed 10 p.p.m. 1248 ad libitum for 8 weeks. The progeny bursa weights were reduced, but the treatment did not interfere with antibody production. PCB treatment had no effect on antibody production after primary or secondary challenges with Brucella abortus. However, after the primary challenge, bursectomized birds of both the control and PCB groups produced significantly fewer antibodies than the nonbursectomized birds. Differential accumulations of the polychlorinated biphenyls occurred in the egg. Chlorinated biphenyls with relative retention times less than that of DDE accumulated to less than one-half of the dietary concentration whereas chlorinated biphenyls with retention times greater than that of DDE accumulated in the egg to a concentration equivalent to the dietary concentration.
1934
S. J. HARRIS, H. C. CECIL, J. BITMAN AND R. J. LILLIE
incubation. Many chicks pipped the shell but failed to hatch. The reasons for the reduction in hatchability remain unknown. The present study reports (1) organ weights of progeny of White Leghorn hens fed several Aroclors and (2) the antibody response of progeny of White Leghorn hens fed Aroclor 1248.
MATERIALS AND METHODS
Immunology (Experiment 2). Two groups of 10 hens each were fed 0 and 10 p.p.m. of Aroclor 1248 in breeder ration for 12 weeks and then were fed the normal ration for the next 7 weeks. Eggs were incubated weekly. Body and organ weights of progeny of hens fed control and Aroclor 1248 feed were taken on Day 1. In each group, 18 of the remaining progeny were divided into three treatment groups: intact, surgically bursectomized, and mock bursectomized. Bursectomies were performed on the day of hatching according to the procedure of Mueller et al. (1960). The chicks were maintained in batteries on a control ad libitum regime. Body weights
EggPCB Residues (Experiment 2). After the hens had been fed PCB for 12 weeks, 15 eggs were randomly selected from both the 10 p.p.m. of Aroclor 1248 and the control groups. The eggs from each treatment were divided into 3 pooled samples containing 5 eggs each and were analyzed for PCB residues. A florisil column was used for clean-up and electron capture gas liquid chromatography was used to determine quantitation (Fries et al., 1973b). A comparison of each peak area in the egg sample with the corresponding peak area in a standard curve of Aroclor 1248 was used to calculate Aroclor 1248 residues. Organ weights were analyzed statistically. Student's " t " test with correction for unequal group size was used (Snedecor, 1956). Chi-
2. Brewer Diagnostic Kits, Brucellosis Card Test, Hynson, Wescott and Dunning, Inc., Baltimore, Maryland 21201.
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Hatchability and Organ Weights (Experiment 1). The White Leghorn hens in the study were the same as those used by Lillie et al. (1975). Individually caged, 29 week old mature White Leghorn hens were divided into 16 groups of 20 each and fed ad libitum a nonmedicated chicken layer breeding ration containing 0, 5, 10, or 20 p.p.m. of Aroclor 1016, 1232, 1242, 1248, and 1254 for 8 weeks followed by the uncontaminated breeder ration for the next 8 weeks. The hens were artificially inseminated biweekly; eggs were stored at 13° C. and 80% humidity, and incubated biweekly. The progeny were raised in floor pens and were fed uncontaminated feed from hatching. Liver, heart, lung, kidney, spleen, and bursa of Fabricius of 1-day- and 4-week-old progeny were weighed.
of all chicks were taken at 1 day and at 3, 6, and 8 weeks of age. The progeny were injected in the median wing vein with 50 x 106 killed Brucella abortus organisms at 8 weeks (primary challenge) and 10 weeks (secondary challenge) of age. Blood was collected from the median wing vein 7 days after each challenge and allowed to clot overnight at room temperature. The serum was heated at 56° C. for 30 minutes and then stored at -20° C. Antisera (0.03 ml.) of individual chicks was tested for agglutination against 0.015 ml. of stained antigen; the Hynson, Wescott and Dunning 2 Brucellosis card test (Nicoletti, 1967) was used. Direct agglutination titers were determined by serial dilution of antisera; the Brucella organism was used as the antigen. The birds were killed at 11 weeks by bleeding from their jugular vein. Body, spleen, heart, bursa of Fabricius, gonad, thymus, and liver weights were taken.
1935
BURSA WEIGHT AND ANTIBODY RESPONSE TO PCBs
TABLE 1.—Hatchability of eggs and bursa of Fabricius and spleen weights of day-old chicks from hens fed Aroclors for 8 weeks (Experiment 1) Progeny organ weights PCB level (p.p.m.)
Hatchability of eggs (percent)
None 1232
0 5 10 20 5 10 20 5 10 20 5 10 20 5 10 20
90 90
1016
1242
1248
1254
7 7
b
51b 83 87 92 87 46" 4
b
86 48 b 6b 98 88 88
n
Bursa mg. ± SE
Spleen mg. ± SE
15 5 5 10 5 5 5 5 10 8 5 10 5 5 5 10
62.7 54.1 59.2 43.7 49.0 46.5 77.8 59.0 40.3 36.3 39.1 42.5 36.3 52.8 48.7 41.2
17.0 17.0 12.4 13.8 15.1 13.8 20.0 16.4 13.6 14.5 16.7 13.5 13.6 15.3 18.1 14.5
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
4.0 4.0 1.5 3.5 b 4.8 3.2 b 2.8 9.6 3.2 b 2.5 b 3.3 b 3.8 b 4.6 b 1.6 4.9 2.9"
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
1.1 0.6 0.8 b 0.8 b 1.1 1.3 0.7 1.0 0.9 b 1.2 2.3 1.0b 2.2" 1.4 1.4 0.7 b
a Last b
two digits of Aroclor designate the % CI, except Aroclor 1016 which contains 41% CI. Significantly different (p < 0.05) from the unsupplemented control when Student's "t" test was used. n = the number of progeny used for determination of mean organ weights. square contingency tests were used to evaluate the immunological data. RESULTS AND DISCUSSION Hatchability and Organ Weights. Lillie et al. (1975) detailed the time course of hatchability failures in this study when 5, 10, and 20 p.p.m. of Aroclor 1232, 1016, 1242, 1248 and 1254 were fed to laying hens. After 8 weeks of feeding, hatchability was severely depressed in eggs from hens fed 10 and 20 p.p.m. of Aroclor 1232,1242, and 1248 (Table 1). Day-old chicks that did hatch from these groups had body, liver, kidney, heart, and lung weights similar to those of the controls (body weight, 39g.; liver, 1.05g.; kidney, 0.245g.; heart, 0.235g.; lung, 0.270g.). However, the spleen and bursa of Fabricius, two organs important in the immune response, were sensitive to PCBs. As hatchability of eggs from hens fed PCBs decreased, the progeny spleen and bursa weights de-
creased (Table 1). Spleen and bursa of day-old chicks from hens fed 10 p.p.m. of Aroclor 1242 and 1248, or 20 p.p.m. of Aroclor 1232, 1242, 1248, and 1254 weighed significantly less (P < 0.05) than those of the controls. Feeding Aroclor 1016 had no effect on hatchability and at only one dosage did Aroclor 1016 have a significant effect; 10 p.p.m. of Aroclor 1016 significantly reduced bursa weight. The effects on hatchability and progeny organ weights are apparently not related to the percentage of chlorine per se because the responses to three Aroclors containing 41 to 48% chlorine (Aroclor 1016, 41% CI; Aroclor 1242, 42% CI, and Aroclor 1248, 48% CI) were not the same. During embryonic development, the bursa appears to be quite sensitive to PCBs. Figure 1 shows the changes in hatchability of eggs from hens fed 20 p.p.m. of Aroclor 1232, 1242, 1248, and 1254 and the bursa weights of day-old and 4-week-old chicks hatched from these eggs. When hatchability de-
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PCB treatment (Aroclor 3 )
1936
S. J. HARRIS, H. C. CECIL, J. BITMAN AND R. J. LILLIE
HATCHABILITY 1 0 0 -„ 80 - . v 60 % 40 \°. 20 i i 0
^jra
"
'X
x-
• \ N" • V /
$ /f
> - *T
1
1
•CONT * 1232 = 1242 ° 1248 • 1254 1 1
BURSA WEIGHT
i
PROGENY)
i
i
i
i
i_
BURSA WEIGHT I ( 4 - W E E K - O L D PROGENY)
1.801.60 GM 1.40 1.20 1.00
2 4 6 8 10 1214 16 U— PCB-s^NO PCB*| WEEKS
FIG. 1. Effects of maternal feeding of 20 p.p.m. PCB and subsequent PCB withdrawal after 8 weeks on hatchability of eggs and bursa of Fabricius weights of progeny. Each point is the mean bursa weight for 5 to 10 progeny. creased, bursa weight decreased. After hens were fed 20 p.p.m. of Aroclor 1232, 1242 or 1248 for 4 weeks, hatchability of their eggs was reduced to 76%, 41%, and 28% respectively. Bursa of Fabricius from these groups also weighed significantly less than control; weights were 62 mg. for control, 50 mg. for Aroclor 1232, 37 mg. for Aroclor 1242, and 38 mg. for Aroclor 1248. Continued feeding of 20 p.p.m. of Aroclor 1232, 1242, 1248, and 1254 for 8 weeks further reduced
In Experiment 2, body, liver, heart, testes and ovarian weights of day-old progeny of Aroclor fed hens were similar to control weights (see Experiment 1). As in Experiment 1, the bursa weights of day-old progeny of hens fed Aroclor 1248 were significantly less than weights of control progeny (Table 2). However, the spleen weights of day-old progeny in Experiment 2 were unaffected until 12 weeks of maternal Aroclor 1248 feeding. When the progeny reached 11 weeks of age, the bursa and spleen weights of treated and control groups did not differ consistently (Table 2). Body, liver, heart, testes and ovarian weights of 11-week-old progeny of Aroclor 1248 and control fed hens were similar (Males: body, 1221g.; liver, 30.Og.; heart, 6.7g.; testes, 2.Og.; spleen, 3.5g.; bursa, 3.0g. Females: body, 940g.; liver, 23.4g.; heart, 4.8g.; ovaries, 0.45g.; spleen, 2.7g.; bursa, 3.6g.). Although increased body weights have been reported for 2-week-old bursectomized chicks (Freeman, 1969; Heller and Perek, 1973), we found no effect of bursectomy on body weights of 3-, 6-, or 11-week-old chicks. Immunology. The bursa is the lymphoid organ responsible for establishing competency in immunoglobulin synthesis. Both chemical and surgical bursectomy interfere with antibody production (Aspinall et al, 1961; Glick et al., 1956; Mueller et al, 1960).
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(DAY-OLD
70-' 6050 MG 40 30 2 0 _i
hatchability, and bursa weights of progeny showed comparable reductions. The effects of the Aroclors were reversible and hatchability and bursa weights returned to control values when the Aroclor feed was replaced with the control feed. In Experiment 2, there was also a steady decline in the hatchability of eggs from hens fed 10 p.p.m. of Aroclor 1248. Hatchability was reduced to 30% of control at 4 weeks and to 9% at 8 weeks. Two weeks after the hens were returned to uncontaminated feed, hatchability returned to normal.
1937
BURSA WEIGHT AND ANTIBODY RESPONSE TO P C B S
TABLE 2.—Bursa of Fabricius and spleen weights of day-old and 11-week-old progeny from hens fed 10 p.p.m. of Aroclor 1248 (PCB) for 12 weeks (Experiment 2) Weight of day-old progeny n
2 4 6 8 10= 12=
15 16 4 5 17 15
92 71d 68d 63b 55e 67e
94 94 80 101 106 85b
2 4
13 20
106 84
101 97
Spleen Bursa (% of control)
n PCB 6 13 3 2 a
Post PCB Feeding 7 93 7 90
— —
— —
— —
— —
91 131
9 6
87 98
127 76d
=All birds that hatched from 10 and 12 weeks PCB treatment were killed for day-old values. Statistical comparison of treated vs. control: b, P < 0.050; c, P < 0.025; d, P < 0.005; e, P < 0.001. n = number of progeny used for determination of mean organ weights. Finding chicks with decreased bursa weights suggested the possibility of partial chemical bursectomy. To determine whether or not progeny immunocompetence was affected by PCBs, we compared antibody production in intact, mock bursectomized and bursectomized progeny of control and treated hens. We reasoned that slight antibody production differences might not be detected in the intact chicks but would be detected in the stressed bursectomized chicks. In Experiment 2 the effects of Aroclor 1248 on the organ and body weights of the progeny were compared with its effect on their immunocompetence. Figure 2 shows the percentage of birds producing antibody 7 days after the primary and secondary challenge with Brucella abortus as determined by the use of Brewer's Card Test. Mock bursectomized chicks from hens fed control or Aroclor 1248 feed showed the same immunological competence as intact birds from these groups. Since there was no effect of operational stress on immunocompetence, data from mock bursectomized and intact birds were combined. Progeny of hens fed PCBs for 8 weeks had an antibody response equal to control progeny after challenge at 8 and 10 weeks with killed Brucella
abortus. Although the day-old (Table 2) and 4-week-old (Fig. 1) progeny of treated hens had lower bursa weights than controls, the 11-week-old progeny of the treated and control hens had similar bursa weights (Table 2). The bursa involutes at 8 to 12 weeks of age when the chicken is reaching sexual maturity and immunological competence is then derived elsewhere. Fewer bursectomized chickens showed antibody responses after primary or secondary challenge, but there was no difference between progeny from control and Aroclor 1248 fed hens (Fig. 2) indicating the importance of the bursa in the immune response and no effect of PCBs on nonbursal immune systems. Because Brewer's Card Testis a qualitative test, agglutination titers were determined by the use of serial dilution. No relationship between the length of time of Aroclor 1248 treatment and the antibody response of their progeny was determined. No statistical difference (chi-square test) in serial dilution titers existed between progeny of the control and those of PCB-fed birds nor between bursectomized and nonbursectomized birds after the primary or secondary challenge. The data failed to indicate that PCB treatment
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Weeks on diet
Weight of 11-week-old progeny Female n Bursa Spleen Bursa Spleen (% of control) (% of control) Feeding 85 125c 14 101 91 9 96 88 117 89 10 68d 89 95 110 113 104 5 78 63 Male
1938
S. J. HARRIS, H. C. CECIL, J. BITMAN AND R. J. LILLIE PRIMARY CHALLENGE 100 r-—r
of hens interfered with antibody production of their progeny. Bursectomy did not elicit complete immunogenic failure although no bursal tissue was found by gross examination at the end of the experiment. In our experiment, bursectomy was not followed by irradiation. Several investigators have proposed that bursal cells travel from the bursa to the bone marrow and then to the spleen and thymus before hatching (Hemmingson and Linna, 1972; Toivanen et al., 1972). Therefore, humoral immunity may have been only partly
As noted above, some organochlorines increase avian susceptibility to disease organisms. However, other investigators have found that feeding organochlorine pesticides (DDT and mirex) to chicks reduced bursa weight but did not interfere with the ability of the chick to produce antibodies after the administration of bovine serum albumin or sheep-red-blood cells (Glick, 1974) or heat killed Salmonella pullorum (Latimer and Siegel, 1974). Our experiment agrees with the studies that demonstrate organochlorines decrease bursal weight but do not suppress antibody response to killed organisms. Although organochlorines do not interfere with antibody production to killed organisms, organochlorines may increase susceptibility to live pathogenic organisms. PCB Residue Accumulation in the Egg. Because the hatchability of eggs from hens fed 10 p.p.m. Aroclor 1248 was greatly reduced,
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FIG. 2. Percentage of White Leghorn progeny with positive antibody responses 7 days after Brucella abortus challenge during 8 weeks of maternal feeding of 10 p.p.m. Aroclor 1248. Intact progeny from hens fed control f e e d ^ H ; Intact progeny from hens fed Aroclor 1 2 4 8 ^ 3 ; Bursectomized progeny from hens fed control feed k^L; Bursectomized progeny from hens fed Aroclor 1248 CV*. a P < 0.005 intact vs. bursectomized. b P < 0.001 intact vs. bursectomized.
reduced by surgical bursectomy. Although we expected immunological suppression in the progeny of hens fed Aroclor 1248, we did not find it. No reports concerning the effects of maternal feeding of organochlorines on the immunological response of progeny have been published. However, several studies concerning the effect of organochlorine pesticides on disease course and the conditioning effect of pesticides for infectious diseases have been published. Radhakrishnan et al. (1972) found that White Leghorn chickens become more susceptible to histomoniasis after oral administration of sublethal doses of p,p'-DDT. The pathological lesions of histomoniasis were correlated with DDT residues in the cecal tissues. DDT pretreatment increased susceptibility of bobwhite quail to histomoniasis (Thompson and Emerman, 1974). PCBs pretreatment increased susceptibility of mallard ducklings to duck hepatitis virus (Friend and Trainer, 1970).
1939
BURSA WEIGHT AND ANTIBODY RESPONSE TO PCBs
TABLE 3.—Accumulation of PCB residues in eggs of hens fed 10 p.p.m. of Aroclor 1248 for 12 weeks GLC retention time (minutes)
10 p.p.m. Aroclor 1248 standard (peak area) 833 3,446 743 16,270 7,120 3,766 2,641 18,570 5,028 N.D. 2,821 928 2,546 2,818 838 172 N.D. 194
PCB in chicken egg b Control 3.4 N.D. 3.5 0.3 0.5 N.D. 0.9 1.1 1.4 N.D. 4.3 N.D. 2.6 2.0 6.1 0.5 N.D. N.D.
PCB (p.p.m.) 2.7 0.1 3.5 1.1 1.9 N.D. 1.3 4.3 3.2 1.9a 9.4 N.D. 12.4 11.7 13.5 2.9 0.7 1.3
PCB minus control -0.7 0.1 0 0.8 1.4 0 0.4 3.2 1.8 1.9 5.1 0 9.8 9.7 7.4 2.4 0.7 1.3
"Calculated on the basis of the area of the 6.88 min. peak. N.D. = not detectable. b Each peak area in the control egg samples and the PCB egg samples were compared with the corresponding peak areas in a standard curve of Aroclor 1248 to calculate the Aroclor 1248 residues in the egg samples. eggs were analyzed to determine whether the pattern of the PCB residue in the egg was significantly different from the pattern of the Aroclor 1248 which the hens had ingested. The Aroclor 1248 added to the feed contained 10 major chlorinated biphenyl chromatographic peaks (Table 3: GLC retention times 2.61, 3.42, 4.31, 4.79, 5.12, 6.16, 6.88, 8.58, 10.82 and 12.63 minutes). If these chlorinated biphenyls accumulated in the egg in amounts equivalent to the dietary level, as do other persistent organochlorines (DDT and DDE), we would expect each chlorinated chromatographic peak in the egg to be equivalent to that in the 10 p.p.m. solution of Aroclor 1248. However, as Fries et al. (1973a) have previously reported, differential accumulation of the biphenyls occurred in the egg. The biphenyls with lower retention times (<7 minutes) have fewer chlorine atoms and were readily metabolized by the chicken and thus
accumulated in the egg to a lesser extent than the higher chlorinated biphenyls. Two of the lower chlorinated biphenyls (retention times 2.61 and 4.79 minutes) were completely metabolized by the chicken and did not accumulate in the egg. Other chlorinated biphenyls with retention times less than those of DDE accumulated to less than one-half of the dietary concentration whereas the chlorinated biphenyls with retention times greater than those of DDE accumulated in the egg to a concentration equivalent to the dietary concentration (Table 3). Eggs from treated hens contained an estimated 2 p.p.m. of a chlorinated biphenyl (retention time = 7.60 minutes) that was not present in the Aroclor 1248 fed. This unidentified biphenyl could result from the metabolism of another chlorinated biphenyl. Because the commercial Aroclors contain such a diverse mixture of polychlorinated biphenyls, it is not possible at this
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2.02 2.61 2.89 3.42 4.31 4.79 5.12 6.16 6.88 7.60 8.58 9.05 10.82 12.63 15.02 17.47 20.15 24.16
Relative retention time (DDE = 100) 23.3 30.1 33.3 39.5 49.8 55.3 59.1 71.1 79.4 87.8 99.1 104.5 124.9 145.8 173.5 201.7 232.6 279.0
1940
S. J. HARRIS, H. C. CECIL, J. BITMAN AND R. J. LILLIE
time to ascribe the toxic effects on hatchability of eggs to a single chlorinated biphenyl or its metabolite. ACKNOWLEDGEMENTS We wish to thank A. J. Guidry for his technical advice on the immunological procedures used in this study.
REFERENCES
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Aspinall, R. L., R. K. Meyer and M. A. Rao, 1961. Effect of various steroids on the development of the bursa of Fabricius in chick embryos. Endocrinology, 68: 944-949. Briggs, D. M., and J. R. Harris, 1973. Polychlorinated biphenyls influence on hatchability. Poultry Sci. 52: 1291-1294. Britton, W. M., and T. M. Huston, 1973. Influence of polychlorinated biphenyls in the laying hen. Poultry Sci. 52: 1620-1624. Carlson, R. W., and R. T. Duby, 1973. Embryotoxic effects of three PCBs in the chicken. Bull. Environ. Contam. Toxicol. 9: 261-266. Cecil, H. C , J. Bitman, R. J. Lillie, G. F. Fries and J. Verrett, 1974. Embryotoxic and teratogenic effects in unhatched fertile eggs from hens fed polychlorinated biphenyls (PCBs): Bull. Environ. Contam. Toxicol. 11: 489-495. Freeman, B. M , 1969. The bursa of Fabricius and adrenal cortical activity in Gallus domesticus. Comp. Biochem. Physiol. 29: 639-646. Friend, M., and D. O. Trainer, 1970. Polychlorinated biphenyl: Interaction with duck hepatitis virus. Science, 170: 1314-1316. Fries, G. F., R. J. Lillie, H. C. Cecil and J. Bitman, 1973a. Retention and excretion of polychlorinated biphenyl residues by laying hens. 165th Meet. Am. Chem. Soc. PEST 52 (Abstract) Dallas, Texas, April 9-13. Fries, G. F., G. S. Marrow, Jr. and C. H. Gordon, 1973b. Long term studies of residue retention and excretion by cows fed a polychlorinated biphenyl (Aroclor 1254). J. Agr. Food Chem. 21: 117-121. Glick, B., 1974. Antibody-mediated immunity in the presence of mirex and DDT. Poultry Sci. 53: 14761485. Glick, B., T. S. Chang and R. G. Jaap, 1956. The bursa of Fabricius and antibody production. Poultry Sci. 35: 224-226. Harris, J. R., 1971. Proceedings of the Interagency Meeting on Polychlorinated Biphenyls. Food and Drug Administration, Health, Education and Wel-
fare, Washington, D.C., August 5, 1971, pp. 74-76. Heller, E. D., and M. Perek, 1973. The effect of bursa Fabricius removal in day-old chicks upon growth performance, blood and spleen characteristics. Poultry Sci. 52: 1065-1068. Hemmingson, E. J., and T. J. Linna, 1972. Ontogenetic studies on lymphoid cell traffic in the chicken. 1. Cell migration from the bursa of Fabricius. Int. Arch. Allergy, 42: 693-710. Latimer, J. W., and H. S. Siegel, 1974. Immune response in broilers fed technical grade DDT. Poultry Sci. 53: 1078-1083. Lillie, R. J., H. C. Cecil, J. Bitman and G. F. Fries, 1974. Differences in response of caged White Leghorn layers to various polychlorinated biphenyls (PCBs) in the diet. Poultry Sci. 53: 726-732. Lillie, R. J., H. C. Cecil, J. Bitman, G. F. Fries and J. Verrett, 1975. Toxicity of certain polychlorinated and brominated biphenyls in caged layer performance. Poultry Sci. 54: 1550-1555. McLaughlin, Jr., J., J. Marliac, M. J. Verrett, M. K. Mutchler and O. G. Fitzhugh, 1963. The injection of chemicals into the fertile egg prior to incubation as a toxicity test. Toxicol. Appl. Pharmacol. 5: 760-771. Mueller, A. P., H. R. Wolfe and R. K. Meyer, 1960. Precipitin production in chicken. XXI. Antibody production in bursectomized chickens and in chickens injected with 19-nortestosterone on the fifth day of incubation. J. Immunol. 85: 172-179. Nicoletti, P., 1967. Utilization of the card test in brucellosis eradication. J. Amer. Vet. Med. Ass. 151: 1778-1783. Pichirallo, J., 1971. PCBs; leaks of toxic substances raises issue of effects, regulation. Science, 173: 899-902. Radhakrishnan, C. V., N. P. Thompson and D. J. Forrester, 1972. Susceptibility of chickens fed p, p'-DDT to histomoniasis. Bull. Environ. Contam. Toxicol. 8: 147-152. Scott, M. L., D. V. Vadehra, P. A. Mullenhoff, G. L. Rumsey and R. W. Rice, 1971. Results of experiments on the effects of PCBs on laying hen performance. Proc. Cornell Nut. Conf. Feed Mfr. pp. 56-64. Snedecor, G. W., 1956. Statistical Methods, Ed. 5. The Iowa State University Press, Ames, Iowa, p. 91. Thompson, N. P., and R. L. Emerman, 1974. Interaction of p,p'-DDT with histomoniasis in bobwhite. Bull. Environ. Contam. Toxicol. 11: 474-482. Toivanen, P., A. Toivanen and R. A. Good, 1972. Ontogeny of bursal function in chickens. III. Immunocompetent cell for humoral immunity. J. Exp. Med. 136: 816-831.
U.S. Department of Agriculture, Agricultural Research Service, Animal Physiology and Genetics Institute, and Nutrition Institute, Beltsville, Maryland 20705 (Received for publication February 17, 1976)
POULTRY SCIENCE 55: 1933-1940, 1976
P
1973; Britton and Huston, 1973; Cecil et al, 1974; Lillie et al, 1974, 1975; Scott et al, 1971) or injecting eggs with Aroclor 1242 or 1254 (Carlson and Duby, 1973; McLaughlin et al, 1963) reduces hatchability of chicken eggs and also reduces growth of progeny (Lillie et al, 1974).
OLYCHLORINATED biphenyls (PCBs) are persistent environmental pollutants of industrial origin which accumulate in the lipid of animal tissues. In 1971, leakage of PCBs into fish meal resulted in the contamination of feed consumed by millions of chickens. Large numbers of chickens and eggs containing excessive PCB residues were condemned (Harris, 1971; Pichirallo, 1971). Commercial chicken breeders observed decreased hatchability of eggs from flocks fed contaminated feed. Since that time, laboratory studies have shown that feeding hens 20 p.p.m. of Aroclor 1 1232, 1242 and 1248 (Briggs and Harris,
In previous studies, we characterized the PCB induced decrease in hatchability, the time of embryonic death, and the nature of the teratogenic effects occurring after feeding chicken hens PCBs (Cecil et al, 1974; Lillie et al, 1974, 1975). Most of the hatchability failure occurred during the last 2 days of
1. The PCBs are marketed in the United States under the trade name Aroclor (Monsanto) and contain a mixture of chlorinated biphenyls varying in chlorine content. Aroclor mixtures are designated by a four digit number, the last two numbers specify the percentage of chlorine in the mixture. One exception, Aroclor 1016 (41% chlorine), is a distillate of Aroclor
1242 from which the higher PCB homologs are removed. Aroclor 1016 contains 0.4% higher homologs while Aroclor 1242 contains 7% of the tetra-, pentaand hexachloro-homologs. Trade names are used solely for the purpose of providing specific information and do not imply endorsement or approval by the U.S. Government over other products not mentioned.
1933
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ABSTRACT In Experiment 1, polychlorinated biphenyls (Aroclors 1016, 1232, 1242, 1248, and 1254) were fed ad libitum to White Leghorn chickens at dietary levels of 5, 10, and 20 p.p.m. for 8 weeks. Hatchability of eggs from hens fed 10 and 20 p.p.m. of Aroclors 1232, 1242, and 1248 was reduced. The weights of the spleen and bursa of Fabricius of day-old chicks from hens fed 10 p.p.m. of Aroclors 1242 and 1248 or 20 p.p.m. of Aroclors 1232, 1242, 1248, and 1254 were less than those of control chicks. At 3 weeks of age these organ weights were still less than those of the control chicks. Body weights were similar to those of controls at hatching, but the 3 week weight gains of progeny from hens fed 20 p.p.m. Aroclors 1242 and 1248 were less than gains of control progeny. After replacement of the PCB diet with uncontaminated feed, hatchability improved within 2 weeks and by 8 weeks was similar to that of the controls. Bursa weight of the progeny was the only parameter still reduced after the hens were fed uncontaminated feed. In Experiment 2, hens were fed 10 p.p.m. 1248 ad libitum for 8 weeks. The progeny bursa weights were reduced, but the treatment did not interfere with antibody production. PCB treatment had no effect on antibody production after primary or secondary challenges with Brucella abortus. However, after the primary challenge, bursectomized birds of both the control and PCB groups produced significantly fewer antibodies than the nonbursectomized birds. Differential accumulations of the polychlorinated biphenyls occurred in the egg. Chlorinated biphenyls with relative retention times less than that of DDE accumulated to less than one-half of the dietary concentration whereas chlorinated biphenyls with retention times greater than that of DDE accumulated in the egg to a concentration equivalent to the dietary concentration.
1934
S. J. HARRIS, H. C. CECIL, J. BITMAN AND R. J. LILLIE
incubation. Many chicks pipped the shell but failed to hatch. The reasons for the reduction in hatchability remain unknown. The present study reports (1) organ weights of progeny of White Leghorn hens fed several Aroclors and (2) the antibody response of progeny of White Leghorn hens fed Aroclor 1248.
MATERIALS AND METHODS
Immunology (Experiment 2). Two groups of 10 hens each were fed 0 and 10 p.p.m. of Aroclor 1248 in breeder ration for 12 weeks and then were fed the normal ration for the next 7 weeks. Eggs were incubated weekly. Body and organ weights of progeny of hens fed control and Aroclor 1248 feed were taken on Day 1. In each group, 18 of the remaining progeny were divided into three treatment groups: intact, surgically bursectomized, and mock bursectomized. Bursectomies were performed on the day of hatching according to the procedure of Mueller et al. (1960). The chicks were maintained in batteries on a control ad libitum regime. Body weights
EggPCB Residues (Experiment 2). After the hens had been fed PCB for 12 weeks, 15 eggs were randomly selected from both the 10 p.p.m. of Aroclor 1248 and the control groups. The eggs from each treatment were divided into 3 pooled samples containing 5 eggs each and were analyzed for PCB residues. A florisil column was used for clean-up and electron capture gas liquid chromatography was used to determine quantitation (Fries et al., 1973b). A comparison of each peak area in the egg sample with the corresponding peak area in a standard curve of Aroclor 1248 was used to calculate Aroclor 1248 residues. Organ weights were analyzed statistically. Student's " t " test with correction for unequal group size was used (Snedecor, 1956). Chi-
2. Brewer Diagnostic Kits, Brucellosis Card Test, Hynson, Wescott and Dunning, Inc., Baltimore, Maryland 21201.
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Hatchability and Organ Weights (Experiment 1). The White Leghorn hens in the study were the same as those used by Lillie et al. (1975). Individually caged, 29 week old mature White Leghorn hens were divided into 16 groups of 20 each and fed ad libitum a nonmedicated chicken layer breeding ration containing 0, 5, 10, or 20 p.p.m. of Aroclor 1016, 1232, 1242, 1248, and 1254 for 8 weeks followed by the uncontaminated breeder ration for the next 8 weeks. The hens were artificially inseminated biweekly; eggs were stored at 13° C. and 80% humidity, and incubated biweekly. The progeny were raised in floor pens and were fed uncontaminated feed from hatching. Liver, heart, lung, kidney, spleen, and bursa of Fabricius of 1-day- and 4-week-old progeny were weighed.
of all chicks were taken at 1 day and at 3, 6, and 8 weeks of age. The progeny were injected in the median wing vein with 50 x 106 killed Brucella abortus organisms at 8 weeks (primary challenge) and 10 weeks (secondary challenge) of age. Blood was collected from the median wing vein 7 days after each challenge and allowed to clot overnight at room temperature. The serum was heated at 56° C. for 30 minutes and then stored at -20° C. Antisera (0.03 ml.) of individual chicks was tested for agglutination against 0.015 ml. of stained antigen; the Hynson, Wescott and Dunning 2 Brucellosis card test (Nicoletti, 1967) was used. Direct agglutination titers were determined by serial dilution of antisera; the Brucella organism was used as the antigen. The birds were killed at 11 weeks by bleeding from their jugular vein. Body, spleen, heart, bursa of Fabricius, gonad, thymus, and liver weights were taken.
1935
BURSA WEIGHT AND ANTIBODY RESPONSE TO PCBs
TABLE 1.—Hatchability of eggs and bursa of Fabricius and spleen weights of day-old chicks from hens fed Aroclors for 8 weeks (Experiment 1) Progeny organ weights PCB level (p.p.m.)
Hatchability of eggs (percent)
None 1232
0 5 10 20 5 10 20 5 10 20 5 10 20 5 10 20
90 90
1016
1242
1248
1254
7 7
b
51b 83 87 92 87 46" 4
b
86 48 b 6b 98 88 88
n
Bursa mg. ± SE
Spleen mg. ± SE
15 5 5 10 5 5 5 5 10 8 5 10 5 5 5 10
62.7 54.1 59.2 43.7 49.0 46.5 77.8 59.0 40.3 36.3 39.1 42.5 36.3 52.8 48.7 41.2
17.0 17.0 12.4 13.8 15.1 13.8 20.0 16.4 13.6 14.5 16.7 13.5 13.6 15.3 18.1 14.5
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
4.0 4.0 1.5 3.5 b 4.8 3.2 b 2.8 9.6 3.2 b 2.5 b 3.3 b 3.8 b 4.6 b 1.6 4.9 2.9"
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
1.1 0.6 0.8 b 0.8 b 1.1 1.3 0.7 1.0 0.9 b 1.2 2.3 1.0b 2.2" 1.4 1.4 0.7 b
a Last b
two digits of Aroclor designate the % CI, except Aroclor 1016 which contains 41% CI. Significantly different (p < 0.05) from the unsupplemented control when Student's "t" test was used. n = the number of progeny used for determination of mean organ weights. square contingency tests were used to evaluate the immunological data. RESULTS AND DISCUSSION Hatchability and Organ Weights. Lillie et al. (1975) detailed the time course of hatchability failures in this study when 5, 10, and 20 p.p.m. of Aroclor 1232, 1016, 1242, 1248 and 1254 were fed to laying hens. After 8 weeks of feeding, hatchability was severely depressed in eggs from hens fed 10 and 20 p.p.m. of Aroclor 1232,1242, and 1248 (Table 1). Day-old chicks that did hatch from these groups had body, liver, kidney, heart, and lung weights similar to those of the controls (body weight, 39g.; liver, 1.05g.; kidney, 0.245g.; heart, 0.235g.; lung, 0.270g.). However, the spleen and bursa of Fabricius, two organs important in the immune response, were sensitive to PCBs. As hatchability of eggs from hens fed PCBs decreased, the progeny spleen and bursa weights de-
creased (Table 1). Spleen and bursa of day-old chicks from hens fed 10 p.p.m. of Aroclor 1242 and 1248, or 20 p.p.m. of Aroclor 1232, 1242, 1248, and 1254 weighed significantly less (P < 0.05) than those of the controls. Feeding Aroclor 1016 had no effect on hatchability and at only one dosage did Aroclor 1016 have a significant effect; 10 p.p.m. of Aroclor 1016 significantly reduced bursa weight. The effects on hatchability and progeny organ weights are apparently not related to the percentage of chlorine per se because the responses to three Aroclors containing 41 to 48% chlorine (Aroclor 1016, 41% CI; Aroclor 1242, 42% CI, and Aroclor 1248, 48% CI) were not the same. During embryonic development, the bursa appears to be quite sensitive to PCBs. Figure 1 shows the changes in hatchability of eggs from hens fed 20 p.p.m. of Aroclor 1232, 1242, 1248, and 1254 and the bursa weights of day-old and 4-week-old chicks hatched from these eggs. When hatchability de-
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PCB treatment (Aroclor 3 )
1936
S. J. HARRIS, H. C. CECIL, J. BITMAN AND R. J. LILLIE
HATCHABILITY 1 0 0 -„ 80 - . v 60 % 40 \°. 20 i i 0
^jra
"
'X
x-
• \ N" • V /
$ /f
> - *T
1
1
•CONT * 1232 = 1242 ° 1248 • 1254 1 1
BURSA WEIGHT
i
PROGENY)
i
i
i
i
i_
BURSA WEIGHT I ( 4 - W E E K - O L D PROGENY)
1.801.60 GM 1.40 1.20 1.00
2 4 6 8 10 1214 16 U— PCB-s^NO PCB*| WEEKS
FIG. 1. Effects of maternal feeding of 20 p.p.m. PCB and subsequent PCB withdrawal after 8 weeks on hatchability of eggs and bursa of Fabricius weights of progeny. Each point is the mean bursa weight for 5 to 10 progeny. creased, bursa weight decreased. After hens were fed 20 p.p.m. of Aroclor 1232, 1242 or 1248 for 4 weeks, hatchability of their eggs was reduced to 76%, 41%, and 28% respectively. Bursa of Fabricius from these groups also weighed significantly less than control; weights were 62 mg. for control, 50 mg. for Aroclor 1232, 37 mg. for Aroclor 1242, and 38 mg. for Aroclor 1248. Continued feeding of 20 p.p.m. of Aroclor 1232, 1242, 1248, and 1254 for 8 weeks further reduced
In Experiment 2, body, liver, heart, testes and ovarian weights of day-old progeny of Aroclor fed hens were similar to control weights (see Experiment 1). As in Experiment 1, the bursa weights of day-old progeny of hens fed Aroclor 1248 were significantly less than weights of control progeny (Table 2). However, the spleen weights of day-old progeny in Experiment 2 were unaffected until 12 weeks of maternal Aroclor 1248 feeding. When the progeny reached 11 weeks of age, the bursa and spleen weights of treated and control groups did not differ consistently (Table 2). Body, liver, heart, testes and ovarian weights of 11-week-old progeny of Aroclor 1248 and control fed hens were similar (Males: body, 1221g.; liver, 30.Og.; heart, 6.7g.; testes, 2.Og.; spleen, 3.5g.; bursa, 3.0g. Females: body, 940g.; liver, 23.4g.; heart, 4.8g.; ovaries, 0.45g.; spleen, 2.7g.; bursa, 3.6g.). Although increased body weights have been reported for 2-week-old bursectomized chicks (Freeman, 1969; Heller and Perek, 1973), we found no effect of bursectomy on body weights of 3-, 6-, or 11-week-old chicks. Immunology. The bursa is the lymphoid organ responsible for establishing competency in immunoglobulin synthesis. Both chemical and surgical bursectomy interfere with antibody production (Aspinall et al, 1961; Glick et al., 1956; Mueller et al, 1960).
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(DAY-OLD
70-' 6050 MG 40 30 2 0 _i
hatchability, and bursa weights of progeny showed comparable reductions. The effects of the Aroclors were reversible and hatchability and bursa weights returned to control values when the Aroclor feed was replaced with the control feed. In Experiment 2, there was also a steady decline in the hatchability of eggs from hens fed 10 p.p.m. of Aroclor 1248. Hatchability was reduced to 30% of control at 4 weeks and to 9% at 8 weeks. Two weeks after the hens were returned to uncontaminated feed, hatchability returned to normal.
1937
BURSA WEIGHT AND ANTIBODY RESPONSE TO P C B S
TABLE 2.—Bursa of Fabricius and spleen weights of day-old and 11-week-old progeny from hens fed 10 p.p.m. of Aroclor 1248 (PCB) for 12 weeks (Experiment 2) Weight of day-old progeny n
2 4 6 8 10= 12=
15 16 4 5 17 15
92 71d 68d 63b 55e 67e
94 94 80 101 106 85b
2 4
13 20
106 84
101 97
Spleen Bursa (% of control)
n PCB 6 13 3 2 a
Post PCB Feeding 7 93 7 90
— —
— —
— —
— —
91 131
9 6
87 98
127 76d
=All birds that hatched from 10 and 12 weeks PCB treatment were killed for day-old values. Statistical comparison of treated vs. control: b, P < 0.050; c, P < 0.025; d, P < 0.005; e, P < 0.001. n = number of progeny used for determination of mean organ weights. Finding chicks with decreased bursa weights suggested the possibility of partial chemical bursectomy. To determine whether or not progeny immunocompetence was affected by PCBs, we compared antibody production in intact, mock bursectomized and bursectomized progeny of control and treated hens. We reasoned that slight antibody production differences might not be detected in the intact chicks but would be detected in the stressed bursectomized chicks. In Experiment 2 the effects of Aroclor 1248 on the organ and body weights of the progeny were compared with its effect on their immunocompetence. Figure 2 shows the percentage of birds producing antibody 7 days after the primary and secondary challenge with Brucella abortus as determined by the use of Brewer's Card Test. Mock bursectomized chicks from hens fed control or Aroclor 1248 feed showed the same immunological competence as intact birds from these groups. Since there was no effect of operational stress on immunocompetence, data from mock bursectomized and intact birds were combined. Progeny of hens fed PCBs for 8 weeks had an antibody response equal to control progeny after challenge at 8 and 10 weeks with killed Brucella
abortus. Although the day-old (Table 2) and 4-week-old (Fig. 1) progeny of treated hens had lower bursa weights than controls, the 11-week-old progeny of the treated and control hens had similar bursa weights (Table 2). The bursa involutes at 8 to 12 weeks of age when the chicken is reaching sexual maturity and immunological competence is then derived elsewhere. Fewer bursectomized chickens showed antibody responses after primary or secondary challenge, but there was no difference between progeny from control and Aroclor 1248 fed hens (Fig. 2) indicating the importance of the bursa in the immune response and no effect of PCBs on nonbursal immune systems. Because Brewer's Card Testis a qualitative test, agglutination titers were determined by the use of serial dilution. No relationship between the length of time of Aroclor 1248 treatment and the antibody response of their progeny was determined. No statistical difference (chi-square test) in serial dilution titers existed between progeny of the control and those of PCB-fed birds nor between bursectomized and nonbursectomized birds after the primary or secondary challenge. The data failed to indicate that PCB treatment
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Weeks on diet
Weight of 11-week-old progeny Female n Bursa Spleen Bursa Spleen (% of control) (% of control) Feeding 85 125c 14 101 91 9 96 88 117 89 10 68d 89 95 110 113 104 5 78 63 Male
1938
S. J. HARRIS, H. C. CECIL, J. BITMAN AND R. J. LILLIE PRIMARY CHALLENGE 100 r-—r
of hens interfered with antibody production of their progeny. Bursectomy did not elicit complete immunogenic failure although no bursal tissue was found by gross examination at the end of the experiment. In our experiment, bursectomy was not followed by irradiation. Several investigators have proposed that bursal cells travel from the bursa to the bone marrow and then to the spleen and thymus before hatching (Hemmingson and Linna, 1972; Toivanen et al., 1972). Therefore, humoral immunity may have been only partly
As noted above, some organochlorines increase avian susceptibility to disease organisms. However, other investigators have found that feeding organochlorine pesticides (DDT and mirex) to chicks reduced bursa weight but did not interfere with the ability of the chick to produce antibodies after the administration of bovine serum albumin or sheep-red-blood cells (Glick, 1974) or heat killed Salmonella pullorum (Latimer and Siegel, 1974). Our experiment agrees with the studies that demonstrate organochlorines decrease bursal weight but do not suppress antibody response to killed organisms. Although organochlorines do not interfere with antibody production to killed organisms, organochlorines may increase susceptibility to live pathogenic organisms. PCB Residue Accumulation in the Egg. Because the hatchability of eggs from hens fed 10 p.p.m. Aroclor 1248 was greatly reduced,
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FIG. 2. Percentage of White Leghorn progeny with positive antibody responses 7 days after Brucella abortus challenge during 8 weeks of maternal feeding of 10 p.p.m. Aroclor 1248. Intact progeny from hens fed control f e e d ^ H ; Intact progeny from hens fed Aroclor 1 2 4 8 ^ 3 ; Bursectomized progeny from hens fed control feed k^L; Bursectomized progeny from hens fed Aroclor 1248 CV*. a P < 0.005 intact vs. bursectomized. b P < 0.001 intact vs. bursectomized.
reduced by surgical bursectomy. Although we expected immunological suppression in the progeny of hens fed Aroclor 1248, we did not find it. No reports concerning the effects of maternal feeding of organochlorines on the immunological response of progeny have been published. However, several studies concerning the effect of organochlorine pesticides on disease course and the conditioning effect of pesticides for infectious diseases have been published. Radhakrishnan et al. (1972) found that White Leghorn chickens become more susceptible to histomoniasis after oral administration of sublethal doses of p,p'-DDT. The pathological lesions of histomoniasis were correlated with DDT residues in the cecal tissues. DDT pretreatment increased susceptibility of bobwhite quail to histomoniasis (Thompson and Emerman, 1974). PCBs pretreatment increased susceptibility of mallard ducklings to duck hepatitis virus (Friend and Trainer, 1970).
1939
BURSA WEIGHT AND ANTIBODY RESPONSE TO PCBs
TABLE 3.—Accumulation of PCB residues in eggs of hens fed 10 p.p.m. of Aroclor 1248 for 12 weeks GLC retention time (minutes)
10 p.p.m. Aroclor 1248 standard (peak area) 833 3,446 743 16,270 7,120 3,766 2,641 18,570 5,028 N.D. 2,821 928 2,546 2,818 838 172 N.D. 194
PCB in chicken egg b Control 3.4 N.D. 3.5 0.3 0.5 N.D. 0.9 1.1 1.4 N.D. 4.3 N.D. 2.6 2.0 6.1 0.5 N.D. N.D.
PCB (p.p.m.) 2.7 0.1 3.5 1.1 1.9 N.D. 1.3 4.3 3.2 1.9a 9.4 N.D. 12.4 11.7 13.5 2.9 0.7 1.3
PCB minus control -0.7 0.1 0 0.8 1.4 0 0.4 3.2 1.8 1.9 5.1 0 9.8 9.7 7.4 2.4 0.7 1.3
"Calculated on the basis of the area of the 6.88 min. peak. N.D. = not detectable. b Each peak area in the control egg samples and the PCB egg samples were compared with the corresponding peak areas in a standard curve of Aroclor 1248 to calculate the Aroclor 1248 residues in the egg samples. eggs were analyzed to determine whether the pattern of the PCB residue in the egg was significantly different from the pattern of the Aroclor 1248 which the hens had ingested. The Aroclor 1248 added to the feed contained 10 major chlorinated biphenyl chromatographic peaks (Table 3: GLC retention times 2.61, 3.42, 4.31, 4.79, 5.12, 6.16, 6.88, 8.58, 10.82 and 12.63 minutes). If these chlorinated biphenyls accumulated in the egg in amounts equivalent to the dietary level, as do other persistent organochlorines (DDT and DDE), we would expect each chlorinated chromatographic peak in the egg to be equivalent to that in the 10 p.p.m. solution of Aroclor 1248. However, as Fries et al. (1973a) have previously reported, differential accumulation of the biphenyls occurred in the egg. The biphenyls with lower retention times (<7 minutes) have fewer chlorine atoms and were readily metabolized by the chicken and thus
accumulated in the egg to a lesser extent than the higher chlorinated biphenyls. Two of the lower chlorinated biphenyls (retention times 2.61 and 4.79 minutes) were completely metabolized by the chicken and did not accumulate in the egg. Other chlorinated biphenyls with retention times less than those of DDE accumulated to less than one-half of the dietary concentration whereas the chlorinated biphenyls with retention times greater than those of DDE accumulated in the egg to a concentration equivalent to the dietary concentration (Table 3). Eggs from treated hens contained an estimated 2 p.p.m. of a chlorinated biphenyl (retention time = 7.60 minutes) that was not present in the Aroclor 1248 fed. This unidentified biphenyl could result from the metabolism of another chlorinated biphenyl. Because the commercial Aroclors contain such a diverse mixture of polychlorinated biphenyls, it is not possible at this
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2.02 2.61 2.89 3.42 4.31 4.79 5.12 6.16 6.88 7.60 8.58 9.05 10.82 12.63 15.02 17.47 20.15 24.16
Relative retention time (DDE = 100) 23.3 30.1 33.3 39.5 49.8 55.3 59.1 71.1 79.4 87.8 99.1 104.5 124.9 145.8 173.5 201.7 232.6 279.0
1940
S. J. HARRIS, H. C. CECIL, J. BITMAN AND R. J. LILLIE
time to ascribe the toxic effects on hatchability of eggs to a single chlorinated biphenyl or its metabolite. ACKNOWLEDGEMENTS We wish to thank A. J. Guidry for his technical advice on the immunological procedures used in this study.
REFERENCES
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